NAD+-dependent sirtuin-family deacylases regulate diverse aspects of cellular and organismal homeostasis. Among these, SIRT5 has remained a somewhat enigmatic protein. SIRT5 is a primarily mitochondrial sirtuin that possesses atypical catalytic properties, removing succinyl, malonyl, and glutaryl groups from lysines on its substrate proteins. Although SIRT5 is the dominant cellular activity that targets these post-translational modifications (PTMs), SIRT5 deficiency in cells and whole animals provokes only very mild phenotypes, particularly under basal conditions. Thus, physiologic functions of SIRT5 and its target PTMs have remained somewhat mysterious. Using shRNA and CRISPR/Cas9-based approaches, we find that the sirtuin SIRT5 is critical for survival of specific cancer types, including melanoma and Ewing sarcoma (EWS). Whole transcriptome profiling demonstrates that SIRT5 affects expression of genes integral to survival and stress responses, including the melanoma lineage gene MITF and the FOXO3A transcription factor. Via metabolite profiling, we have identified defects in one carbon metabolism (1CM) conferred by SIRT5 depletion. 1CM consists of a complex set of biochemical reactions required for generation of the universal methyl donor S- adenosyl methionine (SAM), the co-substrate for histone and DNA methylation. In melanoma and EWS cells depleted for SIRT5, H3K4me3 levels are reduced, a phenotype recapitulated by treatment with a SIRT5 small molecule degrader that we recently developed. Remarkably, SAM reconstitution completely rescues the lethality associated with SIRT5 depletion in melanoma. The long-term goal is to elucidate the roles of SIRT5 in maintaining cellular homeostasis. The objective of this proposal is to elucidate roles for SIRT5 in regulating 1CM and nuclear gene expression. The central hypothesis is that SIRT5 regulates activities of 1CM enzymes to promote SAM generation, H3K4 trimethylation and proper gene expression. The rationale is that 1CM is the source of cellular SAM and other key cellular metabolites, and hence characterization of new mechanisms of 1CM regulation is of fundamental biological importance. The work will take place in the context of three Specific Aims. First, roles of SIRT5 in regulating 1CM will be elucidated mechanistically, using metabolite tracing, biochemistry, and other complementary approaches, focusing initially on the MTHFD1L enzyme as a candidate SIRT5 target. Second, the role of H3K4me3 in mediating the effects of SIRT5 on gene expression will be elucidated, using directed ChIP and global ChIP-seq. Third, the role of SIRT5 in regulating H3K4me3 levels and survival in response to 1C stress will be characterized in normal cells and mice. The application is innovative, in that no literature currently links SIRT5 to 1CM, and most phenotypes of SIRT5 depletion described in the literature are remarkably modest. The work is significant, as elucidation of SIRT5 functions in regulating 1CM will identify new aspects of 1CM biology, as well as potentially illuminating new therapeutic opportunities.